Journal of Steroid Biochemistry & Molecular Biology 84 (2003) 537–542
`
`In vitro and in vivo models for the evaluation of potent inhibitors
`of male rat 17␣-hydroxylase/C17,20-lyase
`I. Duc a,∗
`, P. Bonnet a, V. Duranti a, S. Cardinali a, A. Rivière a, A. De Giovanni a,
`J. Shields-Botella a, G. Barcelo b, N. Adje b, D. Carniato b, J. Lafay b,
`J.C. Pascal b, R. Delansorne a
`a Preclinical R&D Department, Théramex, 6 Avenue Prince Héréditaire, Albert 98000, Monaco
`b Chemical R&D Department, Théramex, 6 Avenue Prince Héréditaire, Albert 98000, Monaco
`
`Received 19 August 2002; accepted 27 January 2003
`
`Abstract
`
`The C17,20-lyase is a key enzyme in the biosynthesis of androgens by both the testes and adrenals. A complete inhibition of this enzyme
`would provide an alternative means of androgen suppression for the treatment of prostatic cancers. In the present study, the inhibitory
`effects of new non-steroidal compounds were tested in vitro on rat C17,20-lyase versus abiraterone, a reference steroidal inhibitor. Their
`activities were also evaluated in vivo on plasma testosterone (T) and luteinizing hormone (LH) levels and on testes, adrenals, seminal
`vesicles (SV) and ventral prostate (VP) weights after 3 days of oral treatment to adult male rats (50 mg/kg per day p.o.).
`Inhibition in the nanomolar range was obtained with TX 977, the lead racemate product in this series, and optimization is ongoing based
`on a slight dissociation observed between its two diastereoisomers, TX 1196-11 (S) and TX 1197-11 (R). These non-steroidal compounds
`(including YM 55208, a reference competitor) proved to be more active in vivo than abiraterone acetate in this model, but the observed
`impact on adrenal weight suggests that the specificity of lyase inhibition versus corticosteroid biosynthesis deserves further investigations
`with this new class of potentially useful agents for the treatment of androgen-dependent prostate cancer.
`© 2003 Elsevier Science Ltd. All rights reserved.
`
`Keywords: Steroidogenesis; Androgen; P450C17; Inhibitor of C17,20-lyase; Rat
`
`1. Introduction
`
`The 17␣-hydroxylase/C17,20-lyase plays a key part in
`the pathways of steroid hormone biosynthesis [1,2]. This
`enzyme catalyses two reactions: 17␣ hydroxylation of C21
`steroids and cleavage of C17,20 bond of C21 steroids. The
`17␣ hydroxylation activity is a required step in cortisol
`biosynthesis whereas the C17,20 bond side chain cleavage
`is essential for the biosynthesis of androgens. The 17␣-
`hydroxylase/C17,20-lyase is a cytochrome P450-dependent
`microsomal enzyme (P450C17), which is expressed in tes-
`ticular and adrenal tissues and catalyses the conversion of
`pregnenolone or progesterone into dehydroepiandrosterone
`(DHEA) or androstenedione ( 4A), respectively, two pre-
`cursors of testosterone (T) [3–7]. The use of effective
`and selective inhibitor of P450C17 appears as a possible
`alternative to orchidectomy or other endocrine therapy,
`
`Corresponding author. Tel.: +377-92-05-08-58;
`∗
`fax: +377-92-05-08-81.
`E-mail address: iduc@theramex.mc (I. Duc).
`
`to lower circulating androgens in patients with prostate
`cancer.
`In the rat and other rodents, 17␣-hydroxylase/C17,20-lyase
`is not expressed in zona fasciculata of adrenal cortex, con-
`sequently corticosterone and not cortisol as in human, is
`the major glucocorticoid. Moreover, the rat P450C17 can
`convert both 17␣-hydroxy-pregnenolone and 17␣-hydroxy-
`progesterone (17OHP) into DHEA and 4A, respectively,
`as compared to human and bovine enzyme that can only
`cleave the C17,20 bond of 17␣-hydroxy-pregnenolone [8,9].
`Even so, due to the lack of availability of human tissue and
`variability among samples, the rat remains, in spite of dif-
`ferences in localization and substrate specificity of this en-
`zyme, a suitable model for the in vitro and in vivo evaluation
`of inhibitory potential of new compounds [10,11].
`The first aim of this study was, to compare in vitro, the
`inhibitory activity of new steroidal and non-steroidal com-
`pounds on C17,20-lyase from rat testis microsomes. 17OHP
`was chosen as substrate, and the inhibitory potential of test
`compounds was determined by measuring the end product
`of the reaction, 4A, by radioimmunoassay (RIA).
`
`0960-0760/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.
`doi:10.1016/S0960-0760(03)00078-5
`
`JANSSEN EXHIBIT 2012
`Mylan v. Janssen IPR2016-01332
`
`

`

`I. Duc et al. / Journal of Steroid Biochemistry & Molecular Biology 84 (2003) 537–542
`tions for storage at −70
`◦
`C. The protein concentration of the
`suspension was determined by the method of Bradford [21]
`with the Bio-Rad protein assay kit.
`
`538
`
`the inhibitory potential of the compounds
`Secondly,
`was evaluated in vivo in adult male rats after 3 days of
`oral treatment. This in vivo model allowed a rapid screen-
`ing of new compounds as compared to the classic 15-
`day model [12]. The weights of androgen-target organs
`such as ventral prostate (VP) and seminal vesicles (SV)
`were measured as well as circulating levels of T and LH.
`The weights of androgen-sensitive organs such as testes
`and adrenal glands were also monitored as an index of
`side-effects. Abiraterone (CB7598), abiraterone acetate
`(CB7630), steroid derivatives [13] and YM 55208 [14], a
`non-steroid compound derived from YM 116 [15], were
`used as references as well as the anti-fungal bifonazole
`[16–18].
`
`2.4. C17,20-lyase activity assay
`
`Microsomes were diluted to a final protein concentra-
`tion of 50 ␮g/ml in the reaction mixture which contained
`0.25 M sucrose, 20 mM Tris–HCl (pH 7.4), 10 mM G6P
`◦
`and 1.2 IU/ml G6PDH. After equilibration at 37
`C for
`10 min, the reaction was initiated by addition of ␤NADP
`to obtain a final concentration of 0.6 mM. Prior to the
`distribution of 600 ␮l of the reaction mixture in each
`tube, test compounds were evaporated to dryness under a
`◦
`stream of nitrogen and then were incubated at 37
`C for
`10 min.
`After incubation with inhibitors, 500 ␮l of the reaction
`mixture was transferred to tubes containing 1 ␮M of the
`enzyme substrate, 17OHP. After a further 10 min incubation,
`tubes were placed on ice and the reaction was stopped by
`addition of 0.1 ml NaOH 1N. Tubes were deep-frozen and
`stored at −20
`◦
`C until assayed for 4A levels.
`A 4A RIA was developed and automated on a mi-
`croplate format in our laboratory using a specific antibody
`against 4A and instructions provided by Biogenesis
`(Poole, England). The separation of free and bound antigen
`was achieved with a dextran-coated charcoal suspension.
`After centrifugation, aliquots of the clear supernatant were
`counted in duplicates in a 1450 MicrobetaPlus liquid scin-
`tillation counter (Perkin-Elmer Instruments, Courtaboeuf,
`France). The 4A concentrations of unknown samples
`were determined from the standard curve. The detection
`limit was 0.5 ng/ml and the within and between assay coef-
`ficients of variation were 10.7 and 17.6%, respectively at an
`assay value of 13 ng/ml. The rate of enzymatic reaction was
`expressed as pmol of 4A formed per 10 min and per mg
`of protein. The value of maximum activity without inhibitor
`(control) was set at 100%. The IC50 values were calculated
`using non-linear analysis from the plot of enzyme activity
`(%) against log of inhibitor concentration (GraphPad Prism,
`version 3.0).
`
`2.5. In vivo assays
`
`Adult male Wistar rats weighing 220–240 g were used.
`All the tested compounds were prepared in distilled water
`with a few drops of Tween 80 (polyoxyethylene sorbitan
`monoleate) and administered by oral route at 10 ml/kg per
`day to animals once daily for 3 days. Control group was
`dosed with vehicle (water-Tween 80) and test compounds
`were given orally at 50 mg/kg per day. On the day following
`the last treatment, animals were anaesthetized with isoflu-
`rane. VP, SV, testes and adrenals were removed, dissected
`and weighed. Arterial blood was withdrawn and collected
`into heparinized tubes. Plasma was stored at −20
`◦
`C until
`required.
`
`2. Materials and methods
`
`2.1. Chemical inhibitors and radioactive steroids
`
`TX 977 (racemate), TX 977-11 (salt of TX 977),
`TX 1196-11 (S-enantiomer of TX 977), TX 1197-11
`(R-enantiomer of TX 977), abiraterone (3␤-hydroxy-
`17-(3-pyridyl)-androsta-5,16-diene),
`abiraterone
`acetate
`(3␤-acetoxy-17-(3-pyridyl)-androsta-5,16-diene), and YM
`55208 (2-(1-(1H-imidazol-1-yl)ethyl)-9H-carbazole) were
`synthesized by Théramex (Monaco). Bifonazole (1-(p,␣-
`diphenylbenzyl)-imidazole) was purchased from Sigma
`(St. Louis, MO, USA). [1,2,6,7-3H]-Androt-4-ene-3,17-
`dione(3H- 4A) was purchased from NEN Life Science
`Products (Boston, MA, USA).
`Other not listed or not specified compounds and reagents
`were from Sigma (St. Quentin Fallavier, France).
`
`2.2. Animals
`
`Adult Wistar male rats were selected for the present study.
`They were purchased from Iffa Credo (Elevage des Oncins,
`L’Arbresle, France). They were housed under conditions of
`12 h light–dark, maintained in an air-conditioned room and
`provided with a standard diet of AO4C pellets from UAR
`(Villemoisson-sur-Orge, France) and filtered mains water ad
`libitum.
`
`2.3. Preparation of rat testis microsomes
`
`Testes from 230 to 310 g rats were obtained by scrotal
`castration. They were washed with an isotonic solution of
`NaCl, pooled, weighed and then blended with an ultra-turrax
`homogenizer in 0.25 M sucrose, 20 mM Tris–HCl (pH 7.4).
`The homogenate was centrifuged at 10 000× g for 30 min at
`C. The supernatant obtained was centrifuged at 100 000×
`◦
`4
`◦
`g for 60 min at 4
`C. The microsomal pellet [19,20] thus ob-
`tained was resuspended in 5 mM MgCl2, 50 mM Tris–HCl,
`pH 7.4 buffer, treated briefly with a dual/kontes homoge-
`nizer to ensure full dispersion and divided into 400 ␮l por-
`
`

`

`I. Duc et al. / Journal of Steroid Biochemistry & Molecular Biology 84 (2003) 537–542
`
`539
`
`2.6. T and LH RIA assays
`
`Plasma was used for the determination of T as described
`in the 125I-T assay kit supplied by Diagnostic Systems Lab-
`oratories Inc. (Webster, TX, USA). The detection limit was
`0.1 ng/ml and the within and between assay coefficients of
`variation were 13.9 and 13.7%, respectively at an assay value
`of 0.5 ng/ml.
`The assay for LH used the rat specific 125I-LH assay
`system from Amersham Biosciences Europe GmbH (Orsay,
`France). The detection limit was 0.8 ng/ml and the within
`and between assay coefficients of variation were 5.2 and
`1.9%, respectively at an assay value of 3.1 ng/ml.
`
`2.7. Statistical analysis
`
`Statistical analysis was performed using the SAS soft-
`ware version 6.12 (SAS Institute, Grégy-Sur-Yerres, FR).
`For IC50, organ weights and plasma hormone levels, homo-
`geneity of variances was checked by Levene’s test. Results
`were analyzed using the parametric analysis of variance
`followed by multiple range tests or the non-parametric
`Kruskall–Wallis analysis and Wilcoxon tests, depending on
`the homogeneity of the variances. A P value less than 0.05
`was considered as statistically significant.
`
`3. Results
`
`3.1. Inhibition of rat testicular C17,20-lyase
`
`As shown in Fig. 1, a concentration-related inhibition
`of the rat testicular C17,20-lyase activity was obtained with
`
`Fig. 1. Inhibition of rat testicular C17,20-lyase activity. Rat testes micro-
`somes were prepared as described in Section 2. C17,20-lyase activity was
`measured after incubation with 1 ␮M of 17OHP and the indicated con-
`centrations of compounds. Each point represents the mean ± S.E.M. of
`four determinations each made in duplicate in individual experiments.
`
`Table 1
`IC50 for the inhibition of lyase activity in rat testis microsomes
`
`Compound
`
`IC50 (nM)
`5.8 ± 0.8a
`Abiraterone
`6.2 ± 0.5
`YM 55208
`6.4 ± 1.0a
`TX 1196-11, S-enantiomer
`8.7 ± 2.0a
`TX 977 racemate
`13.6 ± 3.4
`∗
`TX 1197-11, R-enantiomer
`21.5 ± 5.6
`∗
`Bifonazole
`Means ± S.E.M. for four determinations.
`a NS: P > 0.05.
`∗
`0.01 < P < 0.05 as compared with YM 55208.
`
`all the test compounds. The residual activity expressed as
`the percentage of total activity obtained in presence of test
`compounds, decreased within a range of 115–1% from 1 to
`128 nM. All test compounds were shown to be potent in-
`hibitors of the C17,20-lyase with IC50 values in the nanomo-
`lar range as summarized in Table 1. The decreasing sequence
`of the inhibitory activity was as follows:
`abiraterone ≥ YM 55208 ≥ TX 1196-11(S-enantiomer)
`≥ TX 977(racemate)
`≥ TX 1197-11(R-enantiomer) ≥ Bifonazole.
`The racemate product and its S-enantiomer, were as po-
`tent as abiraterone and YM 55208,
`the steroidal and
`non-steroidal references, respectively. There was a slight
`but not statistically significant difference between the in-
`hibitory potencies of the two diastereoisomers. Bifonazole
`and the R-enantiomer were the less potent compounds in
`this in vitro model.
`
`3.2. Organ weights
`
`As shown in Fig. 2, after 3 days of oral treatment at
`50 mg/kg per day, abiraterone acetate, markedly inhibited
`VP (−14%) and SV weights (−37%) without affecting
`adrenal weight (−7%). YM 55208 induced a more potent
`reduction in VP (−37%) and SV (−48%) but exhibited an
`harmful effect on adrenals by increasing their weight by
`17%. The racemate compound and its S-enantiomer, pro-
`duced similar notable reductions in VP (−24 and −22%,
`respectively) and SV (−42 and −41%, respectively) but the
`S-enantiomer only caused a slight but statistically significant
`increase in adrenal weight (+11%).
`The R-enantiomer caused a marked reduction in VP
`(−24%) which was comparable to that obtained with its two
`related compounds. However, it induced a weaker inhibi-
`tion of SV (−22%), while the increase observed in adrenal
`weight (15%) was not statistically significant. None of the
`tested compounds modified testis weight.
`
`3.3. Plasma hormone levels
`
`Fig. 2 shows that when administered orally at 50 mg/kg
`per day to adult male rat, abiraterone acetate significantly
`
`

`

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`I. Duc et al. / Journal of Steroid Biochemistry & Molecular Biology 84 (2003) 537–542
`
`rise in LH secretion (378%). The racemate product and its
`S-enantiomer induced a 89% inhibition on T secretion and
`increased LH secretion by 240 and 225%, respectively. The
`R-enantiomer significantly inhibited T levels (−63%) and
`increased LH levels (225%) but its effect on plasma T ap-
`peared to be less marked than that of its related compounds.
`
`4. Discussion
`
`In the search of potent inhibitors of C17,20-lyase, the key
`enzyme in androgen biosynthesis, a chemical series of new
`non-steroidal inhibitors was synthesized and tested in vitro
`and in vivo on the rat C17,20-lyase.
`In the in vitro assay reported here, the HPLC product iso-
`lation [22–25] or the classical measurement of the 3H-acetic
`acid released [26–28] were substituted with a RIA of 4A
`produced from the substrate 17OHP. Optimum conditions
`for rat testicular microsome C17,20-lyase activity measure-
`ment with respect to substrate, cofactor, and protein concen-
`trations, as well as time and compatibility with the range of
` 4A in the RIA were determined during preliminary exper-
`iments, and are described in details in Section 2. The activ-
`ity of the control reaction (substrate without enzyme) was
`found to be less than 4% of the enzyme activity. The kinetic
`study of the C17,20-lyase in our conditions indicated that the
`Km was 230 nM and the Vmax was 260 pmol/(min mg) of
`protein (results not shown).
`With this in vitro assay, a chemical series of new
`non-steroidal compounds was investigated for its inhibitory
`potency on rat C17,20-lyase activity (results not shown).
`One of the compounds was isolated as a lead, TX 977 a
`racemate product, and its salt TX 977-11 was used in in
`vivo assays. Its two diastereoisomers, TX 1196-11 (S) and
`TX 1197-11 (R), were then synthesized and evaluated in
`comparison with abiraterone [29] and YM 55208 [14], as
`a steroidal and a non-steroidal references, respectively, and
`the anti-fungal bifonazole [16]. In vitro, a slight difference
`was observed between the two enantiomers, in favor of the
`S-compound. However, the three compounds were as potent
`as abiraterone and YM 55208 with IC50 in the nanomolar
`range, while bifonazole was the less active compound.
`In order to obtain a fast evaluation of the in vivo activ-
`ity of our new compounds, a short model based on a 3-day
`treatment by oral route and a sacrifice 24 h after the last ad-
`ministration was set-up. In this model, the known inhibitory
`effects of abiraterone acetate [30] and YM 55208 [12] on
`VP and SV weights were reproduced. They were directly
`related to the decrease in circulating T levels obtained by
`inhibition of the C17,20-lyase activity. This decrease in T
`levels was associated with a rise in plasma LH, illustrating
`the absence of the negative feedback of T on the pituitary
`gland. Despite this LH increase, which should stimulate the
`testicular androgen production, YM 55208 was able to main-
`tain very low levels of T, below the LOQ of the assay that
`is 0.1 ng/ml. The lead compound and its two enantiomers
`
`Fig. 2. Anti-androgenic activity in the rat. Adult rats were treated as
`described in Section 2. They were treated orally with 50 mg/kg per day
`for 3 days. On the day following the last treatment, ventral prostate
`seminal vesicles (A), testes and adrenal glands (B) were removed and
`weighed. Arterial blood was collected and stored at −20
`◦
`C till T and
`LH assays (C). Each bar represents the mean ± S.E.M. of at least eight
`∗P < 0.05,
`∗∗P < 0.01,
`∗∗∗P < 0.005 as compared with
`determinations.
`control group.
`
`inhibited T secretion (−48%) and in turn increased LH con-
`centration (192%). YM 55208 was shown to totally inhibit
`plasma T secretion (more than −97%) with levels lower
`than the LOQ. This inhibitory effect, more potent than that
`of abiraterone acetate was also associated with a feedback
`
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`

`I. Duc et al. / Journal of Steroid Biochemistry & Molecular Biology 84 (2003) 537–542
`
`541
`
`were shown to be more potent than abiraterone acetate on
`VP weights and T levels. As in vitro, a slight dissociation
`in favor of the S- versus the R-enantiomer was observed for
`SV weights and T levels. No side-effects were observed for
`none of the test compounds on testis (Fig. 2), liver or body
`weights (results not shown).
`However, the S-enantiomer was found to induce an in-
`crease on adrenal weight. This side-effect although weaker
`than that observed with YM 55208 may indicate a lack of
`specificity in inhibition. Then, since in rat the biosynthesis
`of corticosterone does not require the cytochrome P450C17
`enzyme, a selective inhibitor should not have an effect on
`adrenal function and particularly no effect on corticosterone
`levels. In an attempt to explore this effect on adrenals, cor-
`ticosterone levels were measured after a 1-week treatment
`with the S-enantiomer at the dose of 10 mg/kg per day (re-
`sult not shown). There was no statistically significant dif-
`ference observed in corticosterone levels as compared with
`the intact control group, indicating that this new compound
`is probably devoid of inhibitory activity on other enzymes
`involved in corticosterone biosynthesis.
`In conclusion, optimization is ongoing based on the
`nanomolar range inhibition obtained with TX 977-11 the
`lead of our new chemical series of non-steroidal com-
`pounds and the slight dissociation observed between its two
`diastereoisomers, TX 1196-11 (S) and TX 1197-11 (R).
`Together with YM 55208 in our model, they proved to be
`more active in vivo than abiraterone acetate. However, the
`impact observed on adrenal weight suggests that the speci-
`ficity of lyase inhibition versus corticosteroid biosynthesis
`deserve further investigations with this new class of poten-
`tially useful agents for the treatment of androgen-dependent
`prostate cancer. Specificity, pharmacokinetic and long-term
`models (4 weeks at least) are now in progress.
`
`References
`
`[1] W.L. Miller, Molecular biology of steroid hormone synthesis,
`Endocrinol. Rev. 9 (1988) 295–318.
`[2] I. Hanukoglu, Steroidogenic enzymes: structure, function, and role
`in regulation of steroid hormone biosynthesis, J. Steroid Biochem.
`Mol. Biol. 43 (8) (1992) 779–804.
`steroid
`of P450-dependent
`[3] H. Vanden Bossche,
`Inhibitors
`biosynthesis: from research to medical treatment, J. Steroid Biochem.
`Mol. Biol. 43 (8) (1992) 1003–1021.
`of
`[4] D.F.V. Lewis, P. Lee-Robichaud, Molecular modelling
`steroidogenic cytochromes P450 from families CYP11, CYP17,
`CYP19 and CYP21 based on the CYP102 crystal structure, J. Steroid
`Biochem. Mol. Biol. 66 (4) (1998) 217–233.
`[5] W.L. Miller, R.J. Auchus, D.H. Geller, The regulation of 17,20 lyase
`activity, Steroids 62 (1997) 133–142.
`testis
`[6] M. Namiki, M. Kitamura, E. Buczko, M.L. Dufau, Rat
`P-450(17)alpha cDNA: the deduced amino acid sequence, Biochem.
`Biophys. Res. Commun. 157 (2) (1988) 705–712.
`[7] R.J. Auchus, W.L. Miller, Molecular modeling of human P450c17
`(17␣-hydroxylase/17,20-lyase): Insights into reaction mechanisms
`and effects of mutations, Mol. Endocrinol. 13 (7) (1999) 1169–1182.
`I.M. Bird, The role of cytochrome P450 17␣-
`[8] A.J. Conley,
`hydroxylase and 3␤-hydroxysteroid dehydrogenase in the integration
`
`of gonadal and adrenal steroidogenesis via the 5 and 4 pathways
`of steroidogenesis in mammals, Biol. Reprod. 56 (1997) 789–799.
`[9] B.J. Brock, M.R. Waterman, Biochemical differences between rat
`and human cytochrome P450c17 support the different steroidogenic
`needs of these two species, Biochemistry 38 (5) (1999) 1598–1606.
`[10] R.W. Hartmann, M. Hector, B.G. Wachall, A. Palusczak, M. Palzer, V.
`Huch, M. Veith, Synthesis and evaluation of 17-aliphatic heterocycle-
`substituted steroidal inhibitors of 17alpha-hydroxylase/C17-20-lyase
`(P450 17), J. Med. Chem. 43 (23) (2000) 4437–4445.
`[11] I.P. Nnane, K. Kato, Y. Liu, B.J. Long, Q. Lu, X. Wang, Y.-Z. Ling,
`A. Brodie, Inhibition of androgen synthesis in human testicular and
`prostatic microsomes and in male rats by novel steroidal compounds,
`Endocrinology 140 (6) (1999) 2891–2897.
`[12] J. Li, Y. Li, C. Son, P. Banks, A. Brodie, 4-Pregnene-3-one-20␤-
`carboxaldehyde: a potent inhibitor of 17␣-hydroxylase/C17,20-lyase
`and of 5␣-reductase, J. Steroid Biochem. Mol. Biol. 42 (3/4) (1992)
`313–320.
`[13] S.E. Barrie, M. Jarman, G.A. Potter, British Technology Group
`Limited, UK, 17-Substituted steroids useful in cancer treatment, UK
`Patent Applic. 2,265,624 (1993).
`[14] M. Okada, T. Yoden, E. Kawaminami, Y. Shimada, T. Ishihara,
`M. Kudoh, Yamanoushi Pharmaceutical Co. Ltd., Japan. Preparation
`of azole derivatives as steroid 17–20 lyase inhibitors, WO Patent
`9,509,157 (1994).
`[15] Y. Ideyama, M. Kudoh, K. Tanimoto, Y. Susaki, T. Nanya, T.
`Nakahara, H. Ishikawa, T. Yoden, M. Okada, T. Fujikura, H. Akaza,
`H. Shikama, Novel nonsteroidal inhibitor of cytochrome P45017␣
`(17␣-hydroxylase/C17-20 lyase), YM 116, decreased prostatic
`weights by reducing serum concentrations of T and adrenal androgens
`in rats, Prostate 37 (1998) 10–18.
`[16] M. Ayub, M.J. Levell, Inhibition of testicular 17␣-hydroxylase
`and 17,20-lyase but not 3␤-hydroxysteroid dehydrogenase-isomerase
`or 17␤-hydroxysteroid oxidoreductase by ketoconazole and other
`imidazole drugs, J. Steroid Biochem. 28 (5) (1987) 521–531.
`[17] M. Ayub, M.J. Levell, Inhibition of human adrenal steroidogenic
`enzymes in vitro by imidazole drugs including ketoconazole, J.
`Steroid Biochem. 32 (4) (1989) 515–524.
`[18] H. Vanden Bossche, P. Marichal, J. Gorrens, M.-C. Coene, G.
`Willemsens, D. Bellens, I. Roels, H. Moereels, P.A.J. Janssen,
`Biochemical approaches to selective antifungal activity. Focus on
`azole antifungals, Mycoses 32 (Suppl. 1) (1989) 35–52.
`[19] P.B. Kan, M.A. Hirst, D. Feldman, Inhibition of steroidogenic
`cytochrome P-450 enzymes in rat testis by ketoconazole and related
`imidazole anti-fungal drugs, J. Steroid Biochem. 23 (6A) (1985)
`1023–1029.
`testicular 17␣-hydroxy-
`[20] M. Ayub, M.J. Levell, Inhibition of rat
`lase
`and 17,20-lyase
`activities by anti-androgens
`(flutamide,
`hydroxyflutamide, RU23908, cyproterone acetate) in vitro, J. Steroid
`Biochem. 28 (1) (1987) 43–47.
`[21] M.M. Bradford, A rapid and sensitive method for the quantitation
`of ␮g quantities of protein utilizing the principle of protein dye
`binding, Anal. Biochem. 72 (1976) 248–254.
`[22] S.E. Barrie, M.G. Rowlands, A.B. Foster, M. Jarman, Inhibition of
`17␣-hydroxylase/C17-C20 lyase by bifluranol and its analogues, J.
`Steroid Biochem. 33 (6) (1989) 1191–1195.
`[23] M.E. Lombardo, S.I. Hakky, M.K. Hall, P.B. Hudson, A study
`of androgen biosynthesis by the human testis in vitro, J. Steroid
`Biochem. Mol. Biol. 44 (2) (1993) 191–198.
`[24] G.T. Klus, J. Nakamura, J.-S. Li, Y.-Z. Ling, C. Son, J.A.
`Kemppainen, E.M. Wilson, A.M.H. Brodie, Growth inhibition of
`human prostate cells in vitro by novel
`inhibitors of androgen
`synthesis, Cancer Res. 56 (1996) 4956–4964.
`[25] J.-S. Li, Y. Li, C. Son, A.M.H. Brodie, Synthesis and evaluation
`of pregnane derivatives as inhibitors of human testicular 17␣-
`hydroxylase/C17,20-lyase, J. Med. Chem. 39 (1996) 4335–4339.
`[26] S.L. Miller, J.N. Wright, D.L. Corina, M. Akhtar, Mechanistic
`studies on pregnene side-chain cleavage enzyme (17␣-hydroxylase-
`
`

`

`542
`
`I. Duc et al. / Journal of Steroid Biochemistry & Molecular Biology 84 (2003) 537–542
`
`17,20-lyase) using 18O, J. Chem. Soc. Chem. Commun. (1991)
`157–159.
`[27] M. Akhtar, V.C.O. Njar, J.N. Wright, Mechanistic studies on
`aromatase and related C–C bond cleaving P-450 enzymes, J. Steroid
`Biochem. Mol. Biol. 44 (4–6) (1993) 375–387.
`[28] V.C.O. Njar, G.T. Klus, H.H. Johnson, A.M.H. Brodie, Synthesis of
`novel 21-trifluoropregnane steroids: inhibitors of 17␣-hydroxylase/
`17,20-lyase (17␣-lyase), Steroids 62 (1997) 468–473.
`
`[29] S.E. Barrie, G.A. Potter, M. Jarman, M. Dowsett, Highly potent
`inhibitors of human cytochrome P-450(17␣): activity in vitro and in
`vivo, Br. J. Cancer. 67 (Suppl.) (1993) 75.
`[30] S.E. Barrie, G.A. Potter, P.M. Goddard, B.P. Haynes, M. Dowsett, M.
`Jarman, Pharmacology of novel steroidal inhibitors of cytochrome
`P45017␣ (17␣-hydroxylase/C17-20 lyase), J. Steroid Biochem. Mol.
`Biol. 50 (5/6) (1994) 267–273.
`
`